pv92 Alu

Polymerase Chain Reaction (PCR) & pv92 Alu

This is a two-step lab exercise that amplifies students' DNA obtained painlessly from cheek brush samples. Using the thermocycler at the ScienceWorks lab followed by gel electrophoresis, the frequency for the dimorphic pv92 Alu insert is determined for the class. Hardy-Weinberg equilibrium is calculated as well as genetic distance from various population groups using data from around the world. A discussion of introns, exons, and the "out of Africa" hypothesis of human origins is facilitated by the ScienceWorks' staff.

The idea for this lab comes from the DNA Learning Center at Cold Spring Harbor Labs. Their web site includes data from world populations and calculating engines that facilitate Hardy-Weinberg and Genetic Distance calculations. An excellent animation of the PCR reaction can be found on their site at http://www.dnalc.org/ddnalc/resources/animations.html

Their Help files include a description of pv92 Alu which is summarized below.

What is pv92 Alu?

Since the late 1970s, molecular biologists have been fascinated by the presence in eukaryotic DNA of vast amounts of non-coding sequences called introns or intergenic DNA. The function of non-coding, or "junk", DNA is debated. Interestingly, a large portion of intron DNA consists of repeated segments. One such segment is the Alu family of short interspersed repeated elements (SINEs) which are distributed throughout primate genomes. Over the past 65 million years, the Alu sequence has amplified via an RNA-mediated transposition process to a copy number of about 500,000 comprising an estimated 5% of the human genome. Alu sequences are thought to be derived from the 7SL RNA gene which encodes the RNA component of the signal recognition particle that functions in protein synthesis. Alu elements are approximately 300-bp in length and derive their name from a single recognition site for the endonuclease Alu-I located near the middle of the Alu sequence. Approximately 500-2000 Alu SINEs are restricted to the human genome apparently inserted after the divergence of Homo sapiens from other primates. Many of these Alu sequences are dimorphic, that is, either present or not at the specific site. Since the chance of a new insertion at a precise site is vanishingly small, the presence of an Alu sequence at a given site reflects inheritance of that allele. Thus, the gene frequency for a specific Alu insert can be used to compare human population groups and possibly to deduce the migration of our ancestors.

PROCEDURE FOR OBTAINING DNA BY CHEEKBRUSH

(step A is done at school and sample mailed to FBR for processing)

A. Carefully remove the brush from the tube, then brush the inside of your cheek for a full 60 seconds.

Brush 30 seconds on one side, and then 30 seconds on the other.
Use a clock or watch to time yourself.

B. Place brush into a 1.5 ml tube containing 500 uL of 0.09% NaCl. Swirl for 60 seconds.

C. Remove the brush in as dry a condition as possible and throw into the biotrash bag.

D. Label your tube with your assigned number.

E. Centrifuge the 1.5 ml tube with cheek cells and saline to pellet cells.

F. Decant the saline from the tube leaving a small amount of saline overlying the cell pellet.

G. With a micropipette set at 30 uL, pull the cell/saline solution in and out to mix it well.

H. Remove 30 uL of the cell/saline mix and pipet it into a 0.5 ml tube containing 100uL 0.05 N NaOH.

I. Mix by flicking and place the tube into the thermocycler for 10 mins. at 99 degrees to boil cells.

J. Remove 0.5 ml tube and add 25 uL of Tris-HCl with 10% Chelex and mix.

K. Centrifuge to pellet cell debris and Chelex, and remove 50 uL of the supernatant to a 0.5 ml storage tube.

L. Store in the refrigerator until aliquot used for PCR.

PROCEDURE FOR AMPLIFYING pv92 Alu (done at FBR in thermocycler)

A. After centrifuging, an aliquot of cheek cell DNA sample is added to a PCR tube with pv92 Alu primers.

PCR tubes contain Pharmacia "Ready-To-Go Beads" ™ of buffer, deoxynucleotide triphosphates, magnesium chloride and lyophilized Taq polymerase (the thermo-stable DNA polymerase from "Taq" or Thermus aquaticus).

B. The tubes are placed in the thermocycler and cycled 30 times as follows:

Denature DNA: 94^oC for 30 seconds
Anneal primers: 58^oC for 30 seconds
Extend DNA: 72^oC for 30 seconds

PROCEDURE FOR GEL ELECTROPHORESIS AND DETERMINING pv92 Alu GENE FREQUENCY
(done at school or FBR)

A. Cresol red and sucrose are added to PCR product and placed into wells of a 1% agarose gel.

Electrophoresis for 30 minutes at 120 V separates the PCR products. The illustration below shows how PCR amplifies the segment of DNA with or without an Alu insert and what the gel looks like for each possible genotype.

(Illustration is a screen capture from "Gels PC" also available from the DNA Learning Center)

B. Genotypes possible are:

pv92 Alu insert present on both alleles +/+ (lane 1)
pv92 Alu insert present on one allele +/- (lane 3)
pv92 Alu insert absent on both alleles -/- (lane 2)

Utilizing the data obtained from the students' cheek cell DNA, the number of + and - alleles are calculated and the null hypothesis that the group does not differ from the Hardy-Weinberg equilbirium is tested by Chi-Square analysis.
Genetic distance from the gene frequencies of population groups from around the world is calculated by teams of students and mapped on a world atlas. Hypotheses about human migration, and the "out of Africa" hypothesis plus alternatives, are discussed.

A PubMed abstract of Batzer's paper provides Alu gene frequencies from around the world.
The diversity of mutations found in the human disease cystic fibrosis (CF) can be related to the population differences noted in pv92 Alu. This fact has major implications in DNA testing for CF. The CF gene was discovered in 1989 by Lap-Chee Tsui.

To ask a question about this lab, contact by e-mail